The current is kept constant but voltage keeps on increasing. I am not able to figure out why it does happen ?
Since the voltage U=IxR, this reflects changes in the resistance, which may occur during PAGE when the gel composition is altered, or temperature or salt cencentration of the buffer change. You should control These conditions carefully. Further, if the temperature get to hot, the gel may partially "melt", reducing the resistance and thus alter the voltage at constant current.
the changes in the time your gel runs are mainly due to resistance of the buffers (and the gel itself) to current flow, a good way to minimize this effect is preparing buffer stocks (10x) so the ionic strength varies the less possible among experiments (including those to prepare gel and samples)
the gel composition is same. neither temperature nor salt concentration of buffer is altered. still voltage keeps on decreasing. @markus gerhard
There may me many factors which affect the gel run. You may check the Buffer composition, Buffer pH, Voltage, Temperature and the percentage of the Gel.
Consistency in buffer quantity and composition makes a big difference, and although it shouldn't make a difference, running multiple gels at the same time can alter the amount of time PAGE takes
- I once made the mistake of using a Tris buffer previously adjusted to 7.4 to run my gel that required a pH of 8.2 by adding a lot of NaOH. This resulted in a high ionic strength in the buffer and it affected the rate of movement of proteins through the gel. Make sure that you prepare your buffer fresh and adjust to the right pH.
Dear Sharad
Please check the sample is it homogeneous or heterogeneous because small proteins move fast; check the pore size of the gel so filter the gel before use -impurities can cause problem in the run; check the contents of APS AND TEMED because stacking the gel can change voltage; avoid contamination by hand use clean gloves; check the uniformity of temperature; check for any leakage of current at the termini.
good luck
Dr Y K Lahir
I use constant voltage, and with time the required current keeps on increasing. However the time to run the gel is more or less same. Check your buffers' pH and discard if the pH had changed a lot. Make sure you check the pH after bringing the buffers to room temp, if your store them at 4 degree C.
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